1. Field of the Invention
The present invention relates generally to the fields of molecular and cellular biology, nanotechnology, and fluorescence sensors. More particularly, it relates to nanoplatforms for the detection of pH changes.
2. Description of Related Art
Fluorescence imaging has become an important tool in the study of biological molecules, pathways and processes in living cells thanks to its ability to provide spatial-temporal information at microscopic, mesoscopic and macroscopic levels (see, e.g., Tsien, R. Y. Nat. Rev. Mol. Cell Biol. 2003, 4, SS16; Weissleder, R., Nature 2008, 452, 580; Fernandez-Suarez, M., Nat. Rev. Mol. Cell Biol. 2008, 9, 929). Fluorescent reporter molecules can be broadly divided into two categories: intrinsically expressed fluorescent proteins (e.g., GFP) or externally administered fluorescent probes (e.g., synthetic dyes). Fluorescent protein reporters have greatly impacted studies in basic biological sciences by specific labeling of target proteins and live cell imaging of protein functions (see, e.g., Giepmans, B. N. G. Science 2006, 312, 217; Gross, S., Cancer Cell 2005, 7, 5). External imaging probes have been extensively used in various cellular and animal imaging studies.
Recently, activatable imaging probes that are responsive to physiological stimuli such as ionic and redox potentials, enzymatic expressions, and pH have received considerable attention to probe cell physiological processes (see, e.g., de Silva, A. P., Chem. Rev. 1997, 97, 1515; Zhang, J., Nat. Rev. Mol. Cell Biol. 2002, 3, 906; Lee, S., Chem. Commun. 2008, 4250; Kobayashi, H.; Chem. Res. 2010, 44, 83; Lovell, J. F., Chem. Rev. 2010, 110, 2839; Ueno, T., Nat. Methods 2011, 8, 642). Among these stimuli, pH stands out as an important physiological parameter that plays a critical role in both the intracellular (pHi) and extracellular (pHe) milieu (Alberts, B., Molecular Biology of the Cell; 5th ed.; Garland Science: New York, 2008). For example, the pH of intracellular compartments (e.g. endocytic vesicles) in eukaryotic cells is carefully controlled and directly affects many processes such as membrane transport, receptor cycling, lysosomal degradation, and virus entry into cells (Maxfield, F. R., Nat. Rev. Mol. Cell Biol. 2004, 5, 121; Izumi, H., Cancer Treat. Rev. 2003, 29, 541; Nishi, T., Nat. Rev. Mol. Cell Biol. 2002, 3, 94). Recently, dysregulated pH has been described as another hallmark of cancer because cancer cells display a “reversed” pH gradient with a constitutively increased cytoplasmic pH that is higher than the extracellular pH (pHe) (Webb, B. A., Nat. Rev. Cancer 2011, 11, 671).
Although various pH-sensitive fluorescent probes have been reported (Kobayashi, H., Chem. Rev. 2010, 110, 2620; Han, J. Y., Chem. Rev. 2010, 110, 2709), their pH sensitivity primarily arises from ionizable residues with pH-dependent photo-induced electron transfer (PeT) properties to the fluorophores. One potential drawback for these fluorescent agents is their broad pH response (ΔpH˜2) as dictated by the Henderson-Hasselbalch equation (Atkins, P., Physical Chemistry; Oxford University Press, 2009). This lack of sharp pH response makes it difficult to detect subtle pH differences between the acidic intracellular organelles (e.g., <1 pH difference between early endosomes and lysosomes) (Maxfield, F. R., Nat. Rev. Mol. Cell Biol. 2004, 5, 121; Casey, J. R., Nat. Rev. Mol. Cell Biol. 2010, 11, 50) or pHe in solid tumors (6.5-6.9) (Webb, B. A., Nat. Rev. Cancer 2011, 11, 671; Zhang, X., J. Nucl. Med. 2010, 51, 1167.) over normal tissue environment (7.4). Moreover, simultaneous control of pH transition point and emission wavelengths (in particular, in the near IR range) is difficult for small molecular dyes. Recent attempts to develop pH-sensitive fluorescent nanoparticles primarily employ polymers conjugated with small molecular pH-sensitive dyes (Srikun, D., J. Chem. Sci. 2011, 2, 1156; Benjaminsen, R. V., ACS Nano 2011, 5, 5864; Albertazzi, L., J. Am. Chem. Soc. 2010, 132, 18158; Urano, Y., Nat. Med. 2009, 15, 104) or the use of pH-sensitive linkers to conjugate pH-insensitive dyes (Li, C., Adv. Funct. Mater. 2010, 20, 2222; Almutairi, A., J. Am. Chem. Soc. 2007, 130, 444.). These nanoprobe designs also yield broad pH response and lack the ability to fine-tune pH transition point.